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M Tardy

Publications and source records attributed to M Tardy.

At least 37 records · Page 2Linked to original sources
PubMed1991

DBcAMP effect on the expression of GFAP and of its encoding mRNA in astroglial primary cultures.

Short term and chronic dBcAMP effects on the expression of glial fibrillary acidic protein (GFAP) in astroglial primary cultures are investigated. Short (48 h) and long (more than 7 days) treatments with the cAMP derivative induce both cell shape changes and an increase in GFAP immunolabelling. Such effects are only associated with an increase in GFAP and in GFAP-mRNA levels in the long term treatment. These results suggest that the short term effect of dBcAMP induces post-translational modifications of the protein whereas the long term effect is associated with an increase in GFAP mRNA transcription and/or stability.

Animals↗
PubMed1991

In vitro and post-transplantation differentiation of human keratinocytes grown on the human type IV collagen film of a bilayered dermal substitute.

Using human type IV and type I + III collagens and a new, nontoxic cross-linking procedure, we have developed a cell-free bilayered human dermal substitute for organotypic culture and transplantation of human skin keratinocytes. We have studied the formation of the basement membrane, and the differentiation of keratinocytes grown on the type IV collagen layer of this dermal substitute, in vitro and after grafting onto nude mice. These studies demonstrated the formation of essential constituents of the basement membrane in culture: hemidesmosomes and deposition of extracellular matrix on the top of the type IV collagen were observed as early as 6 days after plating of human keratinocytes. Although the keratinocytes formed a well-organized multilayered epithelium, they exhibited limited differentiation when grown submerged in liquid medium. However, the multilayered sheet obtained after 14 days in submerged culture was composed of a regular basal cell layer, several nucleated suprabasal cell layers containing granular cells, and several dense, anucleated cell layers. The grafting experiments have shown a good biocompatibility of the dermal substitute. It is repopulated by fibroblasts, newly synthesized collagen, vessels, and a few mononuclear cells. At Day 14 after grafting, the type IV collagen layer was still present and very dense, and the basement membrane appeared as in culture, with numerous well-structured hemidesmosomes and deposition of extracellular matrix resembling lamina densa. At Day 55 after transplantation, even if the epidermal graft did not exhibit all the characteristics of the normal epidermis in vivo, it was very close to it. At this stage, the basement membrane was complete, with structures clearly indicative of anchoring fibrils. This new dermal substitute offers many advantages. It is stable and easy to handle. Its production is standardized. The oxidation induced by periodic acid led to a nontoxic cross-linked matrix. This dermal substitute is the first one entirely composed of human collagens. The type I + III collagen underlayer is reorganized when grafted. It supports a type IV collagen top layer which offers an excellent substrate for keratinocytes, favors their anchorage, and favors the formation of the basement membrane in vitro. This dermal substitute could be useful for wound coverage or as an in vitro model for toxicological and pharmacological studies.

Basement Membrane↗
PubMed1991

Expression of various microtubule-associated protein 2 forms in the developing mouse brain and in cultured neurons and astrocytes.

A cDNA probe specific to microtubule-associated protein 2 (MAP2) was used to study the expression of the mRNAs encoding the high- and low-molecular-weight MAP2 variants in cultured neurons and astrocytes. The timing and relative abundance of these MAP2 transcripts and of their encoded proteins were also studied in the developing cerebral hemispheres and cerebellum of the mouse. A 9-kb mRNA, known to encode high-molecular-weight MAP2, was expressed in cultured astrocytes, albeit at a lower level than in neurons. The 6-kb transcript, recently shown to encode low-molecular-weight MAP2 (MAP2c), was expressed in neurons and was the predominant MAP2 transcript of the astrocytes. The level of the 9- and 6-kb transcripts decreased at late stages of astroglial and neuronal cell culture. The 9-kb mRNA was detected in the cerebellum and cerebral hemispheres at every developmental stage. Although the levels of this mRNA varied slightly in the cerebral hemispheres, its expression was biphasic in the cerebellum. This might be explained by the differences in timing of development of the various neuronal cell types formed in these two brain areas. The 6-kb transcript was detected only at early developmental stages in the two brain areas. Correlating the temporal expression of the 9-kb mRNA to that of high-molecular-weight MAP2 indicates that the accumulation of this protein is in part regulated at a cytoplasmic level.

Aging↗
PubMed1990

GFAP turnover during astroglial proliferation and differentiation.

The expression and turnover of the glial fibrillary acidic protein (GFAP) were studied in astroglial primary cultures during postnatal proliferation and maturation. 1. Immunocytochemical studies demonstrated that in immature proliferating astrocytes. GFAP was expressed as a filamentous organized crown around the nucleus whereas in the maturating cells, a GFAP labelled network began to radiate throughout the cytoplasm and GFAP was highly expressed in the astroglial processes. 2. GFAP turnover was studied at 3 periods of culture. The decay of radioactivity from prelabelled GFAP was followed from day 4-12 (immature stage), 11-19 (maturing stage) and 21-29 (morphologically differentiated stage). GFAP displayed a biphasic decay kinetic at each considered period. Two pools of GFAP distinctly appeared. The first one was a fast decaying pool with a half life of 16-18 h and of 5-6 days for the stable one. The unstable pool decreased from 70% to 30% of the total incorporated radioactivity from the proliferating stage to the most mature stage, whereas the stable pool increased proportionally.

Animals↗
PubMed1990

Neuron-glia interactions: effect of serotonin on the astroglial expression of GFAP and of its encoding message.

The trophic effect of serotonin on the glial fibrillary acidic protein (GFAP) expression was investigated in rat brainstem astrocytes in primary culture. GFAP immunolabelling decreased and gliofilaments appeared localized in the cytoplasm periphery. GFAP protein level decreased in parallel with a decrease in its encoding message. Serotonin may act as an inhibitor of GFAP expression either on the transcription or on the stability of the GFAP-mRNA.

Animals↗
PubMed1990

Glutamine synthetase modulation in astrocyte cultures of different mouse brain areas.

Astroglial cells from mouse cerebral hemispheres, cerebellum, olfactory bulbs, and medulla oblongata were grown in the presence of either hormones (hydrocortisone, insulin) or cell second messengers (dBcAMP, dBcGMP). Glutamine synthetase (GS) specific activity, GS protein level, and GS translation were investigated under the effect of these factors. Hydrocortisone produced a simultaneous increase in GS translation, GS level, and activity. This increase was observed in the astrocytes cultured from the four brain areas but at a variable magnitude depending on the area. The hydrocortisone effect appeared at the transcriptional level. Inversely, insulin decreased both the GS activity and the in vitro translated GS. This effect was seen only in the olfactory bulbs and the medulla. DBcAMP increased the GS biological activity only in the cerebral hemisphere cultures. It raised, however, the level of translated GS and GS protein in astrocytes from all the areas, suggesting a post-translational effect for intracellular cAMP. DBcGMP only affected GS in the astrocytes from cerebral hemispheres and the medulla modulating either the GS transcription or the messenger RNA stability. These results suggest specific regulation for GS expression, depending on the brain area from which the cells were dissociated or on the astroglial cell population present in these cultures affecting either the transcription, the mRNA stability, or the biological activity of the protein.

Animals↗
PubMed1990

Fibroblast behavior on gels of type I, III, and IV human placental collagens.

Various collagens were extracted and purified from human placenta after partial pepsin digestion. We prepared type III + I (57:43), enriched type I, type III, and type IV collagens on an industrial level, and studied their biological properties with MRC5 fibroblast cells. Using the process of contraction of a hydrated collagen lattice described by Bell, we found tha the contraction rate was dependent on collagen type composition. The contraction was faster and more pronounced with pepsinized type I collagen than with pepsinized type III + I (57:43) collagen; the lowest rate was obtained with the pepsinized type III collagen. Using a new technique of collagen cross-linking, a gel was made with type IV collagen. This cross-linking procedure, based on partial oxidation of sugar residues and hydroxylysine by periodic acid, followed by neutralization, resulted in an increased number of natural cross-link bridges between oxidized and nonoxidized collagen molecules, without internal toxic residues. The fibroblasts were unable to contract type IV/IVox collagen gels. The type IV/IVox collagen gel was transparent and its amorphous ultrastructure lacked any visible striated fibrils. Fibroblast cells exhibited atypical behavior in these type IV/IVox collagen gels as evidenced by optical and electron microscopy. The penetration of fibroblasts could be measured. Fibroblasts penetrated faster in type IV/IVox collagen gels than in untreated type III + I collagen gels. The lowest rate of penetration was obtained with cross-linked type III + I gels. Fibroblast proliferation was similar on untreated or cross-linked type III + I collagen gels and slightly increased on type IV/IVox collagen gels, suggesting that this cross-linking procedure was not toxic.

Cell Division↗
PubMed1990

[Use of human placental type IV collagen in tympanoplasty. Results of initial animal experimentation].

This study presents the use of human type IV collagen film in myringoplasty in dogs. Evaluation criteria were clinical (repeated otoscopies) and histological (histological analysis of cicatrised membranes). This new biomaterial should seem to be a technological advance on temporal bone aponevrosis or perichondria auto-grafts. This study constitutes the necessary preliminary research to any application in man.

Animals↗
PubMed1989

Comparative marker analysis of the ependymocytes of the subcommissural organ in four different mammalian species.

The subcommissural organ (SCO), classified as one of the circumventricular organs, is composed mainly of modified ependymal cells, attributable to a glial lineage. Nevertheless, in the rat, these cells do not possess glial markers such as glial fibrillary acidic protein (GFAP), protein S100, or the enzyme glutamine synthetase (GS). They receive a synaptic 5-HT input and show pharmacological properties for uptake of GABA resembling the uptake mechanism of neurons. In this study, we examine the phenotype of several mammalian SCO (cat, mouse, rabbit) and compare them with the corresponding features of the rat SCO. In all these species, the SCO ependymocytes possess vimentin as an intermediate filament, but never express GFAP or neurofilament proteins. They do not contain GS as do glial cells involved in GABA metabolism, and when they contain protein S100 (rabbit, mouse), its rate is low in comparison to classical glial or ependymal cells. Thus, these ependymocytes display characteristics that differentiate them from other types of glial cells (astrocytes, epithelial ependymocytes and tanycytes). Striking interspecies differences in the capacity of SCO-ependymocytes for uptake of GABA might be related to their innervation and suggest a species-dependent plasticity in their function.

Animals↗
PubMed1989

Developmental expression of the glial fibrillary acidic protein mRNA in the central nervous system and in cultured astrocytes.

The expression of glial fibrillary acidic protein (GFAP)-mRNA during mouse brain development and in astroglial primary cultures has been investigated by using two approaches: Northern-blot evaluation using a specific cDNA probe, and cell-free translation associated with immunoprecipitation. During brain maturation (4-56 days postnatal), the GFAP-mRNA underwent a biphasic evolution. An increase was observed between birth and day 15 (i.e., during the period of astroglial proliferation), which was followed by a decrease until day 56 (i.e., during astroglial cell differentiation). At older stages (300 days), an increase was observed, which might reflect gliosis. During astroglial in vitro development (7-32 days in culture), the GFAP-mRNA showed similar variations. An increase, observed during the period of astroglial proliferation (7-18 days), was followed by a decrease which occurred in parallel to marked changes in cell shape, cell process outgrowth, and the organization and accumulation of gliofilaments. During the same culture period (7-32 days), alpha-tubulin mRNA, which was used as an internal standard, did not vary significantly. These results show that the increase of the GFAP protein and of gliofilaments observed both in vivo and in vitro during astroglial differentiation cannot be ascribed to an accumulation of the GFAP-mRNA. It might be that more than one mechanism regulates the levels of free and polymerized GFAP and of its encoding mRNA.

Animals↗
PubMed1989

[Prevention of AIDS transmission by syringes and needles in France and Africa].

A study made among drug addicts in the Northern suburbs of Paris enables establishing a stabilisation of positive H.I.V. serology prevalence among them, a fact related to the free sale of syringes and needles. In Africa, the nosocomial transmission of H.I.V. by non-sterilisable but reused needles and syringes is demonstrated, and two cases are described. Its importance is certain but must be precise. The prevention of this epidemiological modality must be done by systematic use of reusable and sterilisable injectable material.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Insulin effect on GABA uptake in astroglial primary cultures.

Astroglial cultures from newborn mouse cerebral cortex contain [125I]Insulin binding sites. Binding was specific, reversible, time dependent and reached equilibrium after 45 min. Insulin analogues compete for this [125I]Insulin binding. Incubation of cerebral cortex astroglial cultures with insulin induced a time- and dose-dependent inhibition of the [3H]GABA high affinity uptake. A decrease in the Vmax rather than an effect on the Km was observed. This effect was dose-dependent and effective at 10(-10) M. Autoradiographic observations on the cell monolayer showed the presence of two groups of cells: one which strongly takes up [3H]GABA and consist in smaller GFAP positive process-bearing cells and another group of much flatter and larger GFAP positive cells which uptake was lower. The smaller stellate cells were apparently the most sensitive to insulin effect. These results: 1) confirm the presence of insulin binding sites on astroglial primary cultures, 2) show an effect of insulin on [3H]GABA high affinity uptake of these cells; this effect being optimal on a stellate-like population of astrocytes, and 3) indicate that insulin may interfere in neuromodulation through astroglial signals.

Animals↗
PubMed1988

Glutamine synthetase: a marker of an astroglial subpopulation in primary cultures of defined brain areas.

Primary cultures from various areas of newborn mouse brain were developed and characterized. Enriched astroglial cultures of the cerebral hemispheres, cerebellum, medulla oblongata and olfactory bulbs contained about 80-90% glial fibrillary acidic protein (GFA) immunolabelled cells. These cultures were composed of a majority of flat, 'protoplasmic-like' cells. The aim of this culture model was used: (1) to study glutamine synthetase (GS) activity during in vitro astroglial development; (2) to consider the hydrocortisone effect on GS activity during both growth and maturation periods, and (3) to determine the development of GS immunoreactivity in the cells and eventual GFA and GS expression in these cells. We observed that GS increased during brain maturation in vivo and in vitro, and that addition of hydrocortisone (1 microM, 48 h) to the culture medium induced varying GS activity depending on the developmental stage and the area. In the four areas studied, the number and intensity of GS-immunolabelled cells reached an optimum between 18 and 30 days in vitro. Only about 50-70% of the cell population was GS positive. Double-labelling experiments showed that three groups of cells coexist whatever the considered area. Two expressed both GFA and GS proteins, the last marker at either a low or a high level, and the third was devoid of GS immunoreactivity. Regional differences in GS-specific activity, GS inducibility and GS immunoreactivity exist in the astroglial population, but the factors responsible for these variations are not yet known.

Animals↗
PubMed1987

Large scale use of Spherosil ion exchangers in plasma fractionation.

Spherosil microbeads are spherical and made of porous silica. Their surface is coated with hydrophilic and/or hydrophobic polymers. They are specially designed for the separation of proteins on an industrial scale either by ion exchange or by bioaffinity chromatography. Since 1980, large columns have been used in Institut Mérieux for the purification of placental albumin and several vaccines. Here we describe a chromatographic process for the purification of human albumin and immunoglobulins (IgG) from 25 1 of plasma per cycle. First the plasma was freed of the coagulation factors and then clarified at pH 5.25. The corresponding supernatant was filtered and processed in sterile conditions. Albumin was then purified by ion exchange on 3 successive columns, respectively containing: 6.25 kg of DEAE SPHEROSIL W-1000; 3.5 kg of QMA SPHEROSIL PH-1000; 8 kg of COOH-SPHEROSIL W-1000. The concentration and pH of the buffers were selected to reduce, as much as possible, the total quantity of ion exchangers required per cycle. IgG were then purified from the filtrate of the first of the previous columns. 1 column of 6.25 kg of DEAE SPHEROSIL W-1000 was used at pH 6.8. The selected chromatographic parameters allowed us to demonstrate a total elimination of HBs Ag and HB Virus when voluntarily added to an initial sample (RIA determination and HBV-DNA analysis by molecular hybridization). A second column of a large pore anion exchanger: DEAE SPHEROSIL LP-3000 was added at the end of the IgG purification, as a final security to avoid any risk of transmitting the Hepatitis B virus. The yield and quality of the final products will be presented.

Chromatography, Gel↗
PubMed1986

Differential immunocytochemical staining for glial fibrillary acidic (GFA) protein, S-100 protein and glutamine synthetase in the rat subcommissural organ, nonspecialized ventricular ependyma and adjacent neuropil.

Antibodies raised against glial fibrillary acidic protein (GFA), S-100 protein (S100) and glutamine synthetase (GS) are currently used as glial markers. The distribution of GFA, S100 and GS in the ependyma of the rat subcommissural organ (SCO), as well as in the adjacent nonspecialized ventricular ependyma and neuropil of the periaqueductal grey matter, was studied by use of the immunocytochemical peroxidase-antiperoxidase technique. In the neuropil, GFA, S100 and GS were found in glial elements, i.e., in fibrous (GFA, S100) and protoplasmic astrocytes (S100, GS). The presence of S100 in the majority of the ventricular ependymal cells and tanycytes, and the presence of GFA in a limited number of ventricular ependymal cells and tanycytes confirm the glial nature of these cells. The absence of S100, GFA and GS from the ependymocytes of the SCO, which are considered to be modified ependymal cells, suggests either a non-astrocytic lineage of these cells or an extreme specialization of the SCO-cells as glycoprotein-synthesizing and secreting elements, a process that may have led to the disappearance of the glial markers.

Animals↗